Evolution within coding and noncoding sequences is markedly different. Most proteins are highly conserved, even between distantly related species such as yeast and human. In contrast, cis-regulatory sequences rarely if ever remain conserved between distantly related species. Furthermore, high rates of genetic variation in gene expression have been found within and between species in organisms ranging from yeast to humans. These observations along with other evidence suggest that many of the phenotypic differences that distinguish individuals or species may be caused by changes in gene regulation. However, the identification of cis-regulatory sequences and functional mutations within these sequences remains a major obstacle in understanding the evolution and biological significance of changes in gene expression. We propose to address this issue using Saccharomyces cerevisiae as a model system. First, we will use mapping, complementation and allele replacement experiments to determine whether genetic variation in resistance to pharmacologically active compounds is caused by mutations in coding or noncoding sequences. Second, we will test whether non-neutral patterns of polymorphism within noncoding sequences are indicative of changes in the expression of adjacent genes. Third, we will develop probabilistic models to identify functional changes in cis-regulatory sequences between species and test these predictions by means of gene expression assays. The detection of functional changes in cis-regulatory sequences will not only provide insight into how changes in gene expression have evolved, but will also advance our ability to identify deleterious, potential disease-causing regulatory mutations in humans.